The long term goal of the work is to understand in structural terms the mechanism of action of the molecular tracks (F-actin and microtubules) and motors (myosin, kinesin, dynein) responsible for motility in the cell. Achieving this goal will ultimately require a synthesis of results obtained by two complementary methods: X-ray crystallography will provide detailed atomic information on the individual proteins and is being carried out in other labs. Cryo-EM and image analysis will provide details of the tertiary and quaternary structure and will show how the individual molecules fit together into the working assemblies found in the cell. Described herein is the application of this later approach to acto-myosin systems together with an investigation of the structural basis for dynamic instability in microtubules. Three types of information are essential for building the functional assemblies from the atomic structures of the individual proteins: (i) the molecular envelope of the assembly, (ii) the locations of the individual proteins within the assembly and (iii) the locations of surface markers on specific proteins. Completion of the following experiments, together with data already collected, will provide all three types of information for the rigor complex of F-actin and the myosin head. In addition, unique information on the mechanism of both actin-based and myosin-based regulation will be obtained. Gold-cluster labelling will be used to locate specific surface residues on the myosin head in decorated filaments. The 3-D structures of single-headed HMM-decorated actin and of crystalline arrays of a myosin I will be determined to provide information on the conformation of the light-chain-containing region of the myosin head. To complete our work on the mechanism of thin filament-based regulation, the structure of F-actin complexed with selected regulatory proteins will be determined. To describe conformational changes occurring in myosin-based regulation, the structure of activated and regulated brush border myosin I complexes to F-actin will be compared. Correlation of 3D EM data with the atomic model of F-actin will be carried out in collaboration with Ken Holmes. Preliminary data on microtubules suggest a structural explanation for their dynamic instability. This hypothesis will be tested in simple experiments using non-hydrolyzable GTP analogues.